1. Field of the Invention
The present invention relates to a recombinant Baculovirus having integrated at least a part of cDNA encoding envelope protein of Japanese encephalitis virus into a genome non-essential to proliferation of Baculovirus, a process for efficiently producing envelope protein of Japanese encephalitis virus using said recombinant Baculovirus and a vaccine for Japanese encephalitis virus.
2. Statement of the Prior Art
As a Japanese encephalitis vaccine, there has been hitherto used a vaccine containing inactivated Japanese encephalitis virus as an effective ingredient. The stock solution of inactivated Japanese encephalitis virus vaccine is produced by inoculating Japanese encephalitis virus Nakayama strain found in the National Institute of Health into newborn mice intra-cerebrally, collecting brains from infected mice purifying by the alcohol protamine method and inactivating virus ["Vaccine of Japan", revised second edition, edited by National Institute of Health Alumini Association (published Jan. 20, 1977 by Maruzen Publishing Co., Ltd.)].
In such a vaccine producing process, Japanese encephalitis virus itself must be handled in large quantities so that the handling was extremely highly dangerous to workers in producing vaccines and production costs were also high.
As a vaccine, antigenic protein having viral antigenicity may also be used, not the virus per se. So it has been investigated to produce the antigenic protein in procaryotic cells or eucaryotic cells using recombinant DNA technique. An attempt has also been made to express envelope protein (hereafter sometime referred to as E protein) of Japanese encephalitis virus by genetic manipulation using yeast as a host (Preliminary Abstract of the 34th Meeting of the Japanese Virus Society held in October 1986, page 91). However, expression of natural E protein was unsuccessful.
The present inventors recently succeeded in expressing natural E protein of Japanese encephalitis virus by the recombinant vaccinia virus integrated cDNA encoding E protein into the genome of vaccinia virus used as a live vaccine. Although the recombinant virus is promising as a live vaccine, it involved a problem that its yield was too small to recover the expressed proteins and to use them for the purpose of vaccination or diagnosis.
On the other hand, methods for expressing an exogenous gene in insect cells using an insect viral vector [Japanese Patent Application KOKAI (the term "KOKAI" is used to mean an unexamined application laid open to public inspection) Nos. 37988/1985 and 5787/1986]have been proposed. It is reported that the amount of protein expressed in these methods is markedly higher than that in the other expression systems.
However, Japanese encephalitis virus belonging to the genus Flavivirus of Togaviridae is characterized in that it has a single stranded RNA genome and that a single long polypeptide monocistronically translated from the genome in virus-infected cells undergoes processing (which is used to mean that a single polypeptide is cleaved with protease in cells to form the respective proteins)into capsid protein, membrane protein, E protein and 5 kinds of non-structural proteins; it is thus difficult in its manipulation to integrate cDNA encoding E protein into Baculovirus genome and express E protein. Therefore, no report has yet been made on an example of the expression.
In view of the prior art described above, the present inventors have made extensive investigations on a process for producing E protein of Japanese encephalitis virus in large quantities and as a result, have found that by selecting a region encoding E protein from cDNA prepared from genome RNA of Japanese encephalitis virus, ligating said region encoding E protein with a promoter of Baculovirus and integrating the ligation product into a genome region non-essential for proliferation of Baculovirus, large quantities of Japanese encephalitis virus E protein utilizable as vaccines or diagnostics can be expressed in infected insect cells. Based on this finding, the present invention has come to be accomplished.